Single-cell RNA sequencing (scRNA-seq) technologies have allowed scientists to tease apart the heterogeneity that can be hidden within bulk RNA samples. This technology has revolutionized how scientists study the transcriptome in the context of cancer, immunology, neuroscience, infectious disease, and much more. However, single-cell platforms have been challenging to work with for a variety of reasons. At Fluent BioSciences, our mission to accelerate the understanding of biology and disease by eliminating barriers to single-cell analysis led us to develop an accessible single-cell platform called PIPseq.

PIPseq was designed so that it can be used in every laboratory for scalable, cost-effective scRNA-seq. We recently spoke with Jamie Padilla, Research Scientist II at the University of New Mexico, on her experience with PIPseq and how this platform can accelerate the widespread adoption of single- cell experiments. Padilla, who has been involved in next generation sequencing studies for almost two decades, has been using PIPseq to understand the effects of drug treatment on gene expression in different types of cancer cell lines, mainly ovarian and endometrial cancers.

PIPseq™ Lowers Cost Associated With Single-Cell RNA Sequencing

While single-cell experiments can provide meaningful information that a bulk RNA sample cannot, it has come at a cost. “Single-cell is a newer platform in the next generation sequencing space,” says Padilla. “And right now, most single-cell platforms on the market are still very expensive.” This cost limitation means that many labs are unable to perform single-cell sequencing experiments. Additionally as many single-cell sequencing platforms require microfluidics or flow cytometry to isolate individual cells, many labs aren’t equipped to do so or do not have access to a core facility. Outsourcing these experiments with already complex workflows can make single-cell experiments even more costly.

Compared to the traditional single-cell platforms, the cost of PIPseq is a fraction of the price, starting at $300 per sample depending on the number of cells profiled, without the need for complex equipment and consumables. “This cost-effectiveness allows us to upscale our projects,” says Padilla. “It’ll give us more data for less cost.” 

PIPseq Simplifies Single-Cell Workflows

Sample Preparation – Fewer Cells Required

Traditionally, single-cell studies have relied on complex flow cytometry or microfluidics technologies to sort and separate cells. Padilla says one of the biggest challenges in single-cell studies, aside from cost, is getting enough viable cells using these methods. Flow cytometry, which often requires booking time at a core facility, requires 10^5 -10^6 cells per sample and likely damages a fraction of the cells. This means that cells with rare characteristics could be missed in the downstream analysis. Microfluidics-based methods also have a low capture efficiency with between 7% and 65% of cells introduced to the microfluidics device being captured. Padilla notes that these complexities are eliminated with PIPseq. 

The workflow is user-friendly from start to finish. “I’m able to complete single-cell RNA sequencing projects at my own bench and on my own timelines. I don’t have to worry about sending samples to another facility and waiting for them to run my samples and I don’t have to worry about having enough viable cells,” says Padilla. The PIPseq protocol offers numerous convenient stopping points and places for quality control checks.

User-Friendly Data Analysis

The simple workflows don’t just stop at the bench. Fluent’s data analysis platform, PIPseeker™, makes it easy for scientists to process paired-end reads in FASTQ format into a counts table of barcodes and genes.“Even if you don’t have a strong bioinformatics or data analysis background, it’s very easy to input your files and analyze your data,” says Padilla. PIPseeker is also fast. “I was able to put together several different analyses and within several hours I had all the information I needed,” Padilla says.

An Innovative Single-Cell Platform and Technical Support

“I’ve worked in science for almost two decades and I’ve never met a company that has been as responsive or helpful as the Fluent Bio team has been,” says Padilla, noting how easy it was to get in touch with technical support and customer support teams and how quickly Fluent’s sequencing core returned data to her. In the future, she will be using PIPseq in immunology experiments and plans to use the new PIPseq™ T100 3’ Single Cell RNA kit which can profile up to 100,000 single cells per reaction.

At Fluent BioSciences, we’re excited to hear customer success stories with single-cell RNA Sequencing, from those starting in single-cell to those with long tenure in the single-cell field like Padilla.  “The idea of actually processing single cells versus bulk RNAseq is amazing in its own right. But, to see the progression of single-cell experiments over the years has been even more impressive,” says Padilla. For Padilla, PIPseq enabled her to launch many single-cell experiments using a simple and quick process. “This platform is going to change the way we will be able to do single-cell experiments in the future,” says Padilla.